The present invention relates to a bioreductive process for the efficient preparation of chiral allylic alcohol intermediates which are useful in the asymmetric synthesis of integrin xcex1vxcex23 receptor antagonists. The process comprises an enantioselective 1,2-reduction of a prochiral xcex1,xcex2-unsaturated ketone using a yeast microorganism to afford a chiral allylic alcohol which can be further processed into the desired substituted nonanoic acid derivative, which is useful as an integrin xcex1vxcex23 receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.
The present invention provides an efficient process for the preparation of a chiral allylic alcohol of structural formula I, 
having the (R)-configuration at the stereogenic center marked with an *; wherein R is hydrogen or methyl.
The preparation of compounds of structural formula I in the racemic form was disclosed in U.S. Pat. No. 6,048,861 (Apr. 11, 2000), which is incorporated by reference herein in its entirety. The racemic allylic alcohols disclosed therein were converted in several steps into the desired 3-(pyrimidin-5-yl)- and 3-(2-methyl-pyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-[1,8]-naphthyridin-2-yl)-nonanoic acids, which are useful as integrin xcex1vxcex23 receptor antagonists for the inhibition of bone resorption. The enantiomerically pure forms of the final product were obtained by means of HPLC resolution of the racemic mixture on a chiral solid support. Since only one antipode of the final product is preferred for use as an integrin xcex1vxcex23 receptor antagonist, the achiral process disclosed in U.S. Pat. No. 6,048,861 is inefficient in the sense that equal amounts of the less preferred enantiomer are obtained.
The present invention provides a process for the preparation of a chiral allylic alcohol of structural formula I having the (R)-configuration at the indicated stereogenic center in an efficient enantioselective fashion via bioreduction of a prochiral xcex1,xcex2-unsaturated ketone of structural formula II with a yeast microorganism. 
The present invention is concerned with a process for the preparation of chiral (R)-allylic alcohols of structural formula I. The process utilizes an enantioselective bioreduction with a yeast microorganism under propagation conditions that give rise to enhanced enantioselectivity in the reduction of a prochiral xcex1,xcex2-unsaturated ketone of structural formula II. The chiral (R)-allylic alcohols obtained in this fashion are key intermediates in the asymmetric synthesis of integrin xcex1vxcex23 receptor antagonists, which are useful for inhibiting bone resorption and treating osteoporosis.
The process of the present invention involves the preparation of a chiral allylic alcohol of structural formula I having the (R)-configuration at the indicated stereogenic center by an asymmetric bioreduction reaction involving the incubation of an enone substrate of structural formula II with whole cells of a yeast microorganism in a suitable propagation medium. 
The chiral allylic alcohols of structural formula I of the present invention can be converted in a 3-step sequence of Claisen rearrangement, hydrogenation, and hydrolysis, as described in U.S. Pat. No. 6,048,861, into the final products of structural formula III, which are useful as integrin xcex1vxcex23 receptor antagonists. 
Prior to to filing date of the present application, samples of the microorganisms, Candida chilensis strain MY1708 and Candida schatavii strain MY1831, were deposited at the American Type Culture Collection (ATCC), Manassass, Va. The culture assess designations are ATCC PTA-4078 and 74439, respectively. The deposits will be maintained in the ATCC under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Upon granting of a patent disclosing the deposits, all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed.
The full taxonomic profiles of Candida chilensis and Candida schatavii are detailed in: The Yeasts, A taxonomic study 4th edition (Editors Kurtzman and Fell 1998 Elsevier). The following is a brief description of growth, morphological and taxonomic characteristics of Candida chilensis and Candida schatavii. 
(a) Candida chilensis strain MY 1708 could be propagated on complex growth media including Saboraud Dextrose Broth (SDB, Difco), Yeast Extract/Peptone, and Potato Dextrose Broth. Galactose, glycerol, and glucose were demonstrated to be assimilated carbon sources and either glutamate or ammonium could satisfy nitrogen source requirement when defined yeast basal media were employed. Fermentative growth was possible only with glucose as a carbon source. Growth on SDB agar plates at 25 C. was apparent within 48 hours. Growth at 29 C. was sluggish and no growth at 35 C. could be observed. Colonies were smooth, white to beige, and irregular at edges. Microscopic evaluation indicated ovoidal to longer elliptical forms (average approximately 4 by 4 uM) typically as single units but with some tendency to cluster.
(b) Candida schatavii strain MY1831 could also be propagated on the complex growth media described above for Candida chilensis. Galactose, glycerol, and glucose also served as carbon sources and glutamate or ammonium could be utilized as carbon source. Either glucose or galactose could be used as carbon source to support fermentative growth. Colony formation was evident on SDB medium within 48 hours at 25 C., 29 C., and in contrast to Candida chilensis, 35 C. Colonies were white to cream and shiny. Ovoid to elliptical morphology (average 4 by 4 uM, similar to chilensis) was observed microscopically typically as single cells or in small clusters ( less than 3).
In one embodiment of the enantioselective bioreduction of the present invention, the yeast microorganism is Candida chilensis strain MY1708 or Candida schatavii strain MY1831. In a class of this embodiment, the yeast microorganism is Candida chilensis strain MY1708.
The bioreduction requires growth of the microorganism, such as Candida chilensis or Candida schatavii, in the presence of a suitable propagation medium, such as Yeast media (YM), Sabouraud dextrose broth (SDB), and Yeast nitrogen base (YNB), or a yeast medium as defined in Table 1, for 24-96 hours followed by a period of contact with the ketone substrate (I) under appropriate incubation conditions. The presence of the allylic alcohol product is observed postcharge when solvent-extracted samples are analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). In a second embodiment, the enone substrate of formula II is supplied at a final concentration of 0.5 to 15 grams per liter. The incubation conditions consist of aeration at a temperature of about 22xc2x0 C. to about 30xc2x0 C. at a pH of about 6.5 to about 8.5, and about 5-50 g/L of glucose to regenerate the required cofactor in whole cells.
In another embodiment, the bioreduction of the present invention is carried out in the presence of a carboxylic acid of structural formula IV: 
wherein
xe2x80x9caxe2x80x9d represents a single bond or a double bond;
n is 0, 1, or 2;
R1 is hydrogen, phenyl, or methyl; and
R2 is C1-4 alkyl or aryl wherein aryl is selected from the group consisting of phenyl, naphthyl, biphenyl, pyridyl, furanyl, thienyl, pyrrolyl, benzofuranyl, benzothiophenyl, and indolyl, wherein the aryl group is unsubstituted or substituted with one to five substituents independently selected from halogen, amino, C1-4 alkylamino, di-(C1-4 alkyl)amino, hydroxy, C1-4 alkoxy, and C1-4 alkyl. When xe2x80x9caxe2x80x9d represents a double bond, both the xe2x80x9ccisxe2x80x9d and xe2x80x9ctransxe2x80x9d geometric isomers are intended to be encompassed within the carboxylic acids of formula IV.
In one class of this embodiment, xe2x80x9caxe2x80x9d represents a double bond, n is 0, R1 is hydrogen, and R2 is aryl. In a subclass of this class, R2 is phenyl unsubstituted or substituted with one to two substituents independently selected from halogen, amino, methyl, and methoxy. A specific embodiment is trans-cinnamic acid. In another class of this embodiment, the carboxylic acid of formula IV is added in an amount of about 1 to about 50 molar equivalents of the enone substrate of formula II.
The addition of a carboxylic acid of formula IV to the fermentation mixture affords lower amounts of the saturated alcohol of formula V and saturated ketone of structural formula VI and an increase in the enantioselectivity of the bioreduction. 
The term xe2x80x9c% enantiomeric excessxe2x80x9d (abbreviated xe2x80x9ceexe2x80x9d) shall mean the % major enantiomer less the % minor enantiomer. Thus, an 80% enantiomeric excess corresponds to formation of 90% of one enantiomer and 10% of the other. The term xe2x80x9cenantiomeric excessxe2x80x9d is synonymous with the term xe2x80x9coptical purity.xe2x80x9d
The term xe2x80x9cenantioselectivexe2x80x9d shall mean a reaction in which one enantiomer is produced (or destroyed) more rapidly than the other, resulting in the predominance of the favored enantiomer in the mixture of products.
A biologically pure sample of Candida chilensis strain MY1708 is currently available under the Budapest Treaty in the permanent culture collection of the American Type Culture Collection, 12301 Parklawn Drive in Rockville, Md. 20852, from which it is available under the Accession Number ATCC PTA-4078.
A biologically pure sample of Candida schatavii strain MY1831 is currently available under the Budapest Treaty in the permanent culture collection of the American Type Culture Collection, 12301 Parklawn Drive in Rockville, Md., from which it is available under the Accession Number ATCC 74439.
The following Examples illustrates the preparation of the compounds of formula I and as such are not to be considered as limiting the invention set forth in the claims appended thereto.